In Drosophila, two groups of genes, the Polycomb group (PcG) and the Trithorax group (TrxG) are important for inheritance of the silenced and the active chromatin state, respectively. Regulatory elements called Polycomb group response elements (PREs) are cis-acting DNA sequences required for the recruitment of chromatin-modifying PcG protein complexes. Recently it has been suggested that TrxG proteins act through either the same or overlapping cis-acting sequences as PcG proteins. Our group is working on understanding how PcG and TrxG proteins get recruited to the DNA. Polycomb group response elements (PREs) are DNA elements through which the Polycomb group (PcG) of transcriptional repressors act. Many of the PcG proteins are associated in two protein complexes that repress gene expression by modifying chromatin. Both of these protein complexes specifically associate with PREs in vivo, however, it is not known how they are recruited or held at the PRE. PREs are complex elements, made up of binding sites for many different proteins. Our laboratory has been working to define all the sequences and DNA binding proteins required for the activity of a 181-bp PRE from the Drosophila engrailed gene. Our goal is to define all of the DNA binding proteins required for the activity of this PRE, and to ask whether all PREs are made up of binding sites for the same proteins. Disruption of any of seven different binding sites within this 181-bp PRE disrupts PRE function. The proteins that bind five of these sites are known: Pho/Phol, GAF/Psq, Zeste, Dsp1, and members of the Sp1/KLF family. Our lab identified Pho and Phol and have been studying their function. Pho and Phol are related proteins that bind the same sequence and have some redundant activities. Pho and Phol are required for PcG protein function and for binding of PcG proteins to the DNA. pho, phol double mutant animals have a PcG phenotype clearly indicating they play a central important role in PcG repression. More recently we have been studying the Sp1/KLF family proteins in Drosophila, to try to determine whether a family member plays a central role in PcG protein silencing. This family of proteins encodes transcription factors and has been extensively studied in mammals. There are 20 Sp1/KLF family members in mammals. In Drosophila there are 9 Sp1/KLF family members, and nine of them bind to a site in the 181-bp PRE we study. We derived a consensus-binding site for the Sp1/KLF Drosophila family members and show that this consensus sequence is present in most of the molecularly characterized PREs. These data suggest that one or more Sp1/KLF family members play a role in PRE function in Drosophila. We recently identified a Sp1/KLF family member, Spps, important for PRE activity. Spps encodes a Sp1/KLF family member most highly related to the ubiquitous mammalian transcription factor Sp3. Spps is a ubiquitous protein, expressed at all stages of development. Strikingly, Spps co-localizes with the PcG protein Psc on polytene chromosomes. The co-localization of Spps with Psc is even greater than the co-localization of Pho with Psc. Further, we showed by chromatin immunoprecipitation that Spps binds to both the en and the bxd PREs in S2 cells and larvae. Our recent genome-wide studies confirm the presence of Spps at PREs. We generated a deletion of the Spps transcription unit by homologous recombination and were surprised that Spps mutants live until the pharate adult stage with no obvious PcG phenotype. However Spps mutants suppress PRE-mediated pairing-sensitive silencing, suggesting that Spps does act in PcG repression. Finally, Spps mutations enhance the Pho phenotype, suggesting these two proteins act together to recruit PcG proteins to the DNA. Another important aspect of our work is to ask whether all PREs are alike. In this regard we characterized PREs from the Drosophila invected gene as well as an additional PRE from the engrailed gene. Our work suggests that there is not one way to make a PRE. For example, it was suggested in the literature that two Pho/Phol binding sites are required for PRE function. Our laboratory found that this is true for some PREs, but in the 181-bp PRE, only one Pho/Phol binding siteis necessary for PRE function. This difference has implications on the mechanism of PcG protein complex recruitment to the DNA. The PRE we study is from the Drosophila engrailed gene. engrailed encodes a homeodomain protein that plays an important role in the development of many different parts of the embryo including the formation of the segments, nervous system, head, and gut. It also plays a very important role in the development of the adult, specifying the posterior compartment of each imaginal disk. Accordingly, engrailed is expressed in a very specific and complex manner in the developing organism. The 181-bp engrailed PRE we have been studying is located near the engrailed promoter from 576 to 395bp upstream of the transcription start site. We are interested in determining the role of this PRE in the control of engrailed expression. One of the first things we learned in our studies is that this PRE is redundant with other flanking PREs in the endogenous engrailed gene. There is another strong PRE located from -1100 to -1500 bp and probably other weak PREs nearby. In fact, when we examined the location of Ph and Pho proteins on engrailed DNA by chromatin immunoprecipation (ChIP), we found that they are bound to a 2.5 kb region extending from the engrailed promoter to about 2.5kb upstream. Therefore, it is perhaps not too surprising that a 500bp deletion that includes the 181-bp PRE and flanking sequence did not lead to ectopic engrailed expression. The remaining PREs were apparently sufficient to recruit PcG proteins. However, what was surprising to us was that loss of this DNA lead to a loss of function phenotype, suggesting that this DNA must also play a positive role in the expression of engrailed. Our experiments suggest that there are multiple positive elements either overlapping with or coincident with the PREs. Our work also suggests that PREs can mediate looping, either with distant PREs or with distant enhancers. Our work shows that PREs can either activate or repress transcription, dependent on the context. This is an important result consistent with the idea that PREs are also the site of action of Trithorax group proteins. Finally, we have recently found that sequences required for promoter-enhancer communication are located within or near the engrailed PREs. The regulatory sequences for the engrailed gene extend over a 70 kb region. Our lab has used reporter constructs to find sequences important for expression in stripes, the nervous system, the head, etc. There are discrete regulatory elements located throughout the 70kb region. However, we were not able to locate the enhancer responsible for expression in the imaginal disks, the precursors of the adult structure. Our data suggests that there is no specific enhancer for expression in the imaginal disks. Rather, expression of engrailed in imaginal disk cells is initiated in the embryo, and then, through epigenetic memory, persists in the imaginal disks. We are currently conducting experiments to test this hypothesis.